Microtubule-based machines are responsible for the determination of cell shape, intracellular transport of organelles, chromosome separation during mitosis, and beating of cilia and flagella. Defects in the function of the motor proteins that driv these machines result in changes in cell shape, misplacement of organelles, infertility, chronic respiratory disease, and a wide array of developmental defects. The long-term goal of the proposed research is to understand the mechanisms that regulate the assembly, targeting, and activity of the dynein family of motors. We have identified several new conserved genes that are involved in the assembly and regulation of dynein motors. We will continue to capitalize on the highly ordered structural organization of the flagellar axoneme and the ease of genetic analysis in Chlamydomonas to further characterize these genes and gene products and identify interacting components that regulate dynein activity. Our specific aims are: (1) To characterize two novel complexes, BOP2 and MBO, that coordinate the assembly and activity of the inner dynein arms. We hypothesize that each complex functions as an adaptor to attach specific dynein isoforms to the 96 nm repeat and interconnect the inner dynein arms. We will use proteomic and molecular strategies to analyze the complexes in vitro, genetic analysis and motility assays to test for interactions in vivo, and high resolution structural methods to localiz the subunits in situ (2) To determine the functions and locations of the N-DRC subunits in the nexin link. We hypothesize that four of the N-DRC subunits have regulatory domains that facilitate interactions with the B-tubule and/or the radial spoke/calmodulin spoke complex. We will screen for mutations in these N-DRC subunits to reveal their functions. We will localize them in the nexin link by SNAP-tagging and high resolution cryoET. We will also analyze the pathway of nexin link assembly in vivo. (3) To characterize components that regulates retrograde motor activity and flagellar assembly. We have generated a D1LIC-GFP construct that rescues the null mutation, and we will use this reagent to probe the mechanisms that activate the retrograde motor. We will investigate the functional significance of the subset of proteins that are reduced in the flagellar proteome of a retrograde IFT mutant. In related studies, we will determine the mechanism by which FLA4 facilitates flagellar assembly. We hypothesize that FLA4 is a conserved cytoplasmic factor that regulates the assembly of IFT motors and IFT particles or the loading of cargoes onto the IFT machinery. The studies will provide basic information about the organization of dyneins and associated regulatory components in cilia and flagella. Given the critical roles played by motor proteins and cilia and flagella in a wide range of human diseases, the studies will also have important implications for the development of diagnostic and therapeutic strategies in the treatment of human disease.